Fig. 2. Antisense transcription across the Xist promoter is diminished on X^pA. (A) Positions of the primers used for cDNA synthesis (a,b,c indicated as arrows) and the region amplified by PCR (d,e,f,g,h) are shown relative to the introduced intron at the XhoI site. Primer a, AS90R2; primer b, R1910J; primer c, R371P1. PCR products were amplified using the following primer sets: d, Tsix2F/Tsix2R; e, Xist1175F/Xist1472R; f, 700P2/Xist-6(-)20; g, Xist (-540)F/8692R; h, 8111F/8418R. (B) The presence or absence of the antisense transcription in the region distal to the intron introduced at the XhoI site was examined by strand-specific RT-PCR in undifferentiated ES cells harboring each of the Tsix^pA and Xist^IVS alleles. Product e was amplified on cDNA primed by a. Product f was amplified on cDNA primed by either b or c. cDNA synthesis was carried out in either the presence (+) or absence (-) of reverse transcriptase. (C) The presence or absence of the elongation form of RNA pol II in the region distal to the mpA cassette was analyzed by ChIP assays using a monoclonal antibody (H5) against CTD phosphorylated at Ser2 in undifferentiated XY, X^dcY, X^pAY and X^IVSY ES cells. The relative abundance of the chromatin immunoprecipitated by the antibody to the input was determined by real-time PCR. Although the elongation form of pol II was similarly distributed in the vicinity of the major transcription start site of Tsix (product d) among all types of ES cells, the level of the polymerase in the region downstream of the mpA cassette (product g and h) was significantly diminished in not only X^dcY but also X^pAY ES cells compared with wild-type and X^IVSY ES cells. (D) RT-PCR was carried out on cDNA prepared from total RNA isolated from the placenta at E12.5. Although the proximal region of Tsix was amplified in all cases, the region distal to the mpA was barely amplified in X^pAY and X^pAX, indicating that antisense transcription in the Xist promoter region was efficiently attenuated by the mpA cassette in the placenta of the developing embryos, as expected.